Studies Of The Effect And Mechanisms Of Triptolide On Platinum-resistant Ovarian Cancer Cell Line SKOV3/DDP In Vitro

Objective:This study investigated the effect of triptolide (TPL) in platinum-resistant humanovarian cancer SKOV3/DDP cells′growth and apoptosis in vitro and explored itsunderlying mechanism in order to determine whether TPL is able to overcomechemoresistance and may be an effective treatment for platinum-resistant ovariancancer, either alone or as an adjunct therapy.Method:1. After SKOV3/DDP cells were exposed to varying concentrations of TPL(0-100nM) for24-48h, cell viability was assessed by MTT assay.2. SKOV3/DDP cells were cultured for24h with varying doses of TPL (0-100nM). The rate of apoptosis was measured by flow cytometry using Annexin V/PIstaining.3. Cells were incubated with100nM TPL for the indicated times (1,12and24h)and stained with H2DCFDA. Flow cytometry was used to detect the intracellular ROSlevel.4. Cells were incubated with100nM TPL or5mM NAC alone for24h orpreincubated with5mM NAC for1h followed by incubation with100nM TPL for24h, intracellular ROS level and apoptosis were measured by flow cytometry.5. SKOV3/DDP cells were cultured for24h with varying doses of TPL (0-100nM). The expression of NF-κB/p65, Bcl-2, caspase3protein was determined byWestern blot.Result:1. SKOV3/DDP cells were treated with various dose (12.5,25,50,100nM) TPLfor24、48h, the equal cell viability was95.192%±0.048%、87.106%±0.379%、65.943%±0.112%、56.426%±0.285%、77.165%±0.228%、64.313%±0.656%、34.452%±0.798%、24.824%±1.450%, respectively. 2. SKOV3/DDP cells were exposed to various dose (12.5,25,50,100nM) TPLfor24h, the equal apoptotic rate was12.53%±0.42%、22.87%±0.31%、35.47%±0.16%、52.19%±0.27%, respectively.3. SKOV3/DDP cells were treated with100nM TPL for1h、12h、24h, thelevel of ROS was increased3.49±0.21fold、7.72±0.42fold、9.17±0.76fold comparedto control.4.5mmol/L NAC had no effect on TPL-induced apoptosis and ROS generationin SKOV3/DDP cells. After treatment of100nM TPL for24h in SKOV3/DDP cells,the level of ROS was increased9.41±0.57fold compared to control and the apoptoticrate was48.2%±1.2%. After SKOV3/DDP cells were pretreatment with5mM NACfor1h and then exposed to100nM TPL for24h, the level of ROS was increased2.72±0.35fold compared to control and the apoptotic rate was21.6%±1.2%.5. Western Blot showed that with the concentration of TPL increasing,expression of NF-κB p65and Bcl-2were reduced and activated caspase3raiseddramatically in SKOV3/DDP cells.Conclusion:1. TPL could inhibit proliferation and induce apoptosis of platinum-resistantSKOV3/DDP cells.2. TPL increased the level of ROS in a dose-and time-dependent manner.3. Pretreatment with N-acetyl-L-cysteine (NAC) abolished the TPL-inducedROS generation and cell apoptosis in SKOV3/DDP cells, indicated that the apoptosisinducing effect of TPL is associated with ROS generation.4. The pro-apoptotic activity of TPL in SKOV3/DDP cells is related to inhibitionof NF-κB, down-regulation of anti-apoptotic protein Bcl-2and activation of caspase3.